US9988436B2 - Composite comprising antibody capable of binding to intranuclear protein of influenza virus - Google Patents

Composite comprising antibody capable of binding to intranuclear protein of influenza virus Download PDF

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US9988436B2
US9988436B2 US15/649,279 US201715649279A US9988436B2 US 9988436 B2 US9988436 B2 US 9988436B2 US 201715649279 A US201715649279 A US 201715649279A US 9988436 B2 US9988436 B2 US 9988436B2
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antibody
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amino acid
acid sequence
influenza virus
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Emina IKEUCHI
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Panasonic Intellectual Property Management Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • the present invention relates to a composite comprising an antibody capable of binding to an intranuclear protein of an influenza virus.
  • the present invention also relates to a detection device and a detection method using the composite.
  • Patent Literature 1 discloses antibodies each capable of binding to an influenza virus. At least a part of the antibodies disclosed in Patent Literature 1 are derived from an alpaca. Patent Literature 1 is incorporated herein by reference.
  • the present invention is a composite, comprising:
  • the present invention provides a novel antibody capable of binding to an intranuclear protein of an influenza virus.
  • the present invention also provides a detection device and a detection method using the composite comprising the novel antibody.
  • FIG. 1A is a map of a vector used to ligate various genes included in a gene library of a VHH antibody.
  • FIG. 1B shows the detail of the vector map shown in FIG. 1A .
  • FIG. 2 is a vector map used to express the VHH antibody.
  • FIG. 3A is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration: 0.195 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
  • FIG. 3B is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration: 0.39 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
  • FIG. 3C is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration: 0.78 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
  • FIG. 3D is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration: 1.56 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
  • FIG. 3E is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration:3.125 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
  • FIG. 3F is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration: 6.25 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
  • FIG. 3G is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration: 12.5 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
  • FIG. 4A is a graph showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type A H1N1 11675.
  • FIG. 4B is a graph showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type A H1N1 40205.
  • FIG. 4C is a graph showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type A H2N2.
  • FIG. 4D is a graph showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type A H3N2.
  • FIG. 4E is a graph showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type A H7N9.
  • FIG. 4F is a graph showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type B.
  • the antibody according to the present invention is capable of binding to an influenza virus type A.
  • the antibody according to the present invention is capable of binding to an intranuclear protein of an influenza virus type A.
  • an antibody capable of binding to an influenza virus consists of, in an N- to C-direction, the following structural domains. N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C where
  • FR denotes a framework region amino acid sequence and CDR denotes a complementary determining region amino acid sequence.
  • the CDR1 consists of an amino acid sequence represented by RTIFNPNVMG (SEQ ID NO: 01).
  • the CDR2 consists of an amino acid sequence represented by DISLSGSTNYADSVKG (SEQ ID NO: 02).
  • the CDR3 consists of an amino acid sequence represented by NAISGAPGRY (SEQ ID NO: 03).
  • the CDR1, the CDR2, and the CDR3 are represented by SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03 respectively.
  • the FR1, the FR2, the FR3, and the FR4 consist of amino acid sequences represented by QVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 04), WYRQAPGKQRELVA (SEQ ID NO: 05), RFTISRDNAKNTMYLQMNSLKPEDTAVYYCNT (SEQ ID NO: 06), and WGQGAQVTVSS (SEQ ID NO: 07), respectively.
  • the antibody according to the present invention consists of the following amino acid sequence.
  • the antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 does not exhibit antigen cross reactivity with regard to an influenza virus, such as an influenza virus type B, other than the influenza virus type A.
  • the antibody according to the present invention is employed in a detection device or in a detection method for detecting the intranuclear protein of an influenza virus type A.
  • the antibody according to the present invention is used in a state where the antibody is bound to at least one selected from the group consisting of a solid support and a labeled substance.
  • a shape and a material of the solid support is not limited.
  • An example of the shape of the solid support is a plate, a bead, a disk, a tube, a filter, and a film.
  • An example of a material of the solid support is a polymer such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, or polymethylmethacrylate, a metal such as gold, silver, or aluminum, or glass.
  • a known method such as a physical adsorption method, a covalent binding method, an ion bonding method, or a cross-linking method is employed as a method for binding the antibody to the solid support.
  • a labeled substance such as a fluorescent substance, a luminescent substance, a dye, and a radioactive substance is used.
  • a known method such as a physical adsorption method, a covalent binding method, an ion bonding method, or a cross-linking method is employed as a method for binding the antibody to the labeled substance.
  • the composite including the antibody is brought into contact with an analyte. Then, detected is a change of a physical amount based on an antigen-antibody reaction of the intranuclear protein of the influenza virus type A contained in the analyte and the antibody included in the composite.
  • a physical amount is, for example, luminescence intensity, chromaticity, light transmission, turbidness, absorbance, or radiation dose.
  • a known method such as an enzyme immunoassay method, an immunochromatography method, a latex agglutination method, a radioimmunoassay method, a fluorescence immunoassay method, or a surface plasmon resonance spectroscopy method is employed as an example of the detection method.
  • the detection device in which the antibody according to the present invention is employed includes a detector for detecting any one of the physical amount which may be changed on the basis of the antigen-antibody reaction.
  • the detector is composed of a known device such as a photometer, a spectroscope, or a dosimeter.
  • VHH antibodies capable of binding to an intranuclear protein included in an influenza virus type A H1N1 were prepared in accordance with the following procedures.
  • the intranuclear protein may be referred to as “NP”.
  • an alpaca was immunized using a recombinant intranuclear protein (SEQ ID NO: 24) derived from an Influenza virus type A H1N1 (A/Puerto Rico/8/34/Mount Sinai) as an antigen.
  • the recombinant intranuclear protein was prepared using a Brevibacillus expression system by Higeta Shoyu Co., Ltd. The recombinant intranuclear protein was adjusted with an adjuvant before administrated to an alpaca.
  • the recombinant intranuclear protein having a concentration of 100 micrograms/milliliter was administered to the alpaca.
  • the recombinant HA protein having the same concentration was administered to the alpaca, again.
  • the alpaca was immunized with the recombinant HA protein five times for five weeks.
  • blood of the alpaca was extracted. Then, mononuclear cells were acquired from the blood as below.
  • a blood cell separation solution (available from COSMO BIO Co., Ltd., trade name: Lymphoprep) was added to a lymphocyte separation tube (available from Greiner Bio-One Co., Ltd., trade name: Leucosep). Then, the solution was subject to centrifugation under a temperature of 20 Celsius degrees at 1,000 ⁇ g for one minute.
  • the blood extracted from the alpaca was treated with heparin. Then, an equivalent amount of phosphate buffered saline (hereinafter, referred to as “PBS”) was added to the thus-treated blood to obtain a sample solution. Then, the sample solution was added to the lymphocyte separation tube to which the blood cell separation solution was added.
  • PBS phosphate buffered saline
  • the lymphocyte separation tube was subject to centrifugation under a temperature of 20 Celsius degrees at 800 ⁇ g for thirty minutes.
  • the fraction containing the mononuclear cells was collected. PBS having three times volume was added. The fraction was subject to centrifugation under a temperature of 20 Celsius degrees at 300 ⁇ g for five minutes. The precipitate was suspended with PBS mildly. After the suspending, 10 microliters of the suspension was separated in order for the count of the number of the cell. The remaining suspension was subject to centrifugation under a temperature of 20 Celsius degrees at 300 ⁇ g for five minutes.
  • RNA storage solution (trade name: RNAlater) having a volume of 2 milliliters was added to the precipitate. Then, the solution was suspended mildly. The suspension was injected to two tubes each having a volume of 1.5 milliliters. Each tube included 1 milliliter of the suspension. The tube was stored under a temperature of ⁇ 20 Celsius degrees. The suspension (5 microliters) separated for the count of the number of the cell was mixed with a Tork's solution (15 microliters), and the number of the mononuclear cells was counted with a counting chamber.
  • RNA was extracted from the mononuclear cells, and a cDNA gene library of VHH antibody was formed in accordance with the following procedure. In the following procedure, RNase-free-grade reagents and instruments were used.
  • RNA isolation reagent (Trade name: TRIzol Regent, 1 milliliter) was added to the mononuclear cell fraction. The reagent was mixed mildly with the fraction, and left at rest at room temperature for five minutes. Chloroform (200 microliters) was added to the reagent, and the reagent was shaken strongly for fifteen seconds. The reagent was left at a room temperature for two-three minutes. The reagent was subject to centrifugation under a temperature of 4 Celsius degrees for 15 minutes at 12,000 or less x g.
  • the supernatant was moved to a new tube.
  • RNase-free water and chloroform 200 microliters, each) were added to the tube.
  • 500 milliliters of isopropanol was added to the tube.
  • the liquid included in the tube was stirred with a vortex mixer. The liquid was left at rest at room temperature for ten minutes. Then, the liquid was subject to centrifugation under a temperature of 4 Celsius degrees for fifteen minutes at 12,000 or less x g. The supernatant was removed, and the precipitate was rinse with one milliliter of 75% ethanol. This solution was subject to centrifugation under a temperature of four Celsius degrees for five minutes at 7,500 or less x g. The solution was dried to obtain total RNA. The obtained total RNA was solved in RNase-free water.
  • a kit including a reverse transcriptase was employed.
  • the kit was available from Takara Bio Inc., as a trade name of PrimeScript II 1 st strand cDNA Synthesis Kit.
  • the Random 6 mer and Oligo dT primer included in the kit were used as primers.
  • the cDNA was obtained in accordance with the standard protocol attached in the kit.
  • the gene of the VHH antibody included in the alpaca was obtained from the cDNA by a PCR method.
  • An enzyme for PCR was available from Takara Bio Inc., as a trade name of Ex-taq.
  • the mixture solution was subject to the following PCR method.
  • the mixture solution was heated at temperature of 95 Celsius degrees for two minutes.
  • This cycle was repeated thirty times.
  • the following primers were used in the present PCR method.
  • Primer 1 5′ - GGTGGTCCTGGCTGC -3′ (SEQ ID NO: 10)
  • Primer 2 5′ - ctgctcctcgcGGCCCAGCCGGCCatggcTSAGKT GCAGCTCGTGGAGTC -3′ (SEQ ID NO: 11)
  • Primer 3 5′ - TGGGGTCTTCGCTGTGGTGCG -3′ (SEQ ID NO: 12)
  • Primer 4 5′ - TTGTGGTTTTGGTGTCTTGGG -3′ (SEQ ID NO: 13)
  • Primer 5 5′ - tttgCtctGCGGCCGCagaGGCCgTGGGGTCTTCG CTGTGGTGCG -3′
  • Primer 6 5′ -tttgCtctGCGGCCGCagaGGCCgaTTGTGGTTTTG GTGTCTTGGG-3′
  • a primer set A composed of the cDNA, Primer 1 and Primer 3 and a primer set B composed of the cDNA, Primer 1 and Primer 4 were used.
  • a primer set C composed of the gene amplified with the primer set A, Primer 2, and Primer 3
  • a primer set D composed of the gene amplified with the primer set B, Primer 2, and Primer 4.
  • a primer set E composed of the gene amplified with the primer set C, Primer 2, and Primer 5
  • a primer set F composed of the gene amplified with the primer set D, Primer 2, and Primer 6.
  • the gene library of the VHH antibody was formed.
  • the gene library of the VHH antibody included the genes amplified with the primer sets E and F.
  • phase I library was formed from the gene library of the VHH antibody in accordance of the following procedures.
  • a plasmid Vector 1 (4057 bp, see FIG. 1A ) derived from a commercially available plasmid pUC119 (for example, available from Takara Bio. Inc.,) was treated with a restriction enzyme SfiI.
  • the restriction enzyme site SfiI(a) consists of the gene sequence represented by GGCCCAGCCGGCC (SEQ ID NO: 15).
  • the restriction enzyme site SfiI(b) consists of the gene sequence represented by GGCCTCTGCGGCC (SEQ ID NO: 16).
  • FIG. 1B shows a detail vector map of the plasmid Vector 1.
  • the plasmid Vector 1 consists of the following gene sequence.
  • VHH antibody gene fragments were obtained.
  • the thus-treated plasmid Vector 1 was mixed with the VHH antibody gene fragments at a ratio of 1:2.
  • An enzyme (available from Toyobo Co. Ltd., trade name: Ligation High ver. 2) was injected to the mixture solution.
  • the mixture solution was left at rest at a temperature of 16 Celsius degrees for two hours. In this way, each of the VHH anti body gene fragments was ligated into the plasmid Vector 1.
  • Coli bacteria (available from Takara Bio. Inc., trade name: HST02) were transfected with the thus-ligated plasmid Vector 1.
  • the coli bacteria were incubated for fifteen hours on a 2YT plate culture medium containing ampicillin at a concentration of 100 micrograms/milliliter. In this way, obtained was a library of phases each of which displays a protein obtained from the gene fragment included in the gene library of the VHH antibody.
  • the library of the phases had a concentration of 5 ⁇ 10 7 /milliliter.
  • VHH antibodies capable of specifically binding to the intranuclear protein were obtained from the phage library in accordance with the following procedures.
  • Coli bacteria (HST02) to which the VHH antibody gene fragment included in the gene library of the VHH antibody was introduced were incubated at temperature of 30 Celsius degrees in the 2YT AG culture medium containing 100 micrograms/milliliter of ampicillin and 1% glucose in such a manner that a value OD 600 indicating absorbance reached 1.0.
  • the 2YT AG culture has a volume of 100 milliliters. In this way, the Coli bacteria were proliferated.
  • Helper phages (available from Invitrogen company, trade name: M13K07) were added to the coli bacteria culture medium in such a manner that the multiplicity of infection was approximately twenty.
  • the culture medium was warmed for about thirty minutes at temperature of 37 Celsius degrees. Then, the culture medium was subject to centrifugation at rotation speed of 4000 rpm for ten minutes to collect the coli bacteria. The coli bacteria was incubated overnight at temperature of 30 Celsius degrees in a 2YTAK culture medium containing 100 micrograms/milliliter of ampicillin and 1% 50 micrograms/milliliter of kanamycin, while subjected to centrifugation at 213 rpm.
  • the incubation liquid (100 milliliters) containing the thus-incubated coli bacteria were injected to two centrifugation tubes(volume: 50 milliliters, each).
  • the two centrifugation tubes were subject to centrifugation for ten minutes at rotation speed of 4,000 rpm. Then, the supernatants (20 milliliters, each) were collected.
  • the supernatants (40 milliliters) were added to a 20% polyethylene glycol solution (10 milliliters) containing NaCl (2.5M). Then, the mixture solution was mixed upward down. Subsequently, the mixture solution was cooled on an ice for approximately one hour. The mixture was subject to centrifugation for ten minutes at rotation speed of 4,000 rpm. Then, the supernatant was removed. PBS containing 10% glycerol was injected toward the precipitate. Finally, the precipitate was loosened and dissolved. In this way, a library of phases each of which displays the VHH antibody was obtained.
  • NP was mixed with PBS to prepare an NP solution.
  • concentration of NP was 2 micrograms/milliliter.
  • the NP solution (2 milliliters) was injected into an immunotube (available from NUNC Co. Ltd.). The NP solution was left at rest in the immunotube for one hour. In this way, NP was immobilized in the immunotube.
  • the inside of the immunotube was filled with PBS which contained 3% skim milk (available from Wako Pure Chemical Industries, Ltd.). In this way, NP was blocked as an antigen in the immunotube.
  • the immunotube was left at rest at room temperature for one hour. Subsequently, the inside of the immunotube was washed three times with PBS.
  • the library of the phages each of which displays the VHH antibody (Concentration: approximately 10E+11/milliliter) was mixed with 3 milliliters of PBS containing 3% skim milk to prepare a mixture solution.
  • the mixture solution was injected into the immunotube in which the NP antigen was immobilized.
  • the immunotube was provided with a lid formed of a parafilm. Then, the immunotube was rotated upward down in a rotator for ten minutes.
  • the immunotube was left at rest at room temperature for one hour.
  • PBST PBS containing 0.05% tween 20
  • the inside of the immunotube was filled with PBST. Subsequently, the immunotube was left at rest for ten minutes. Then, the inside of the immunotube was washed ten times with PBST.
  • the immunotube was provided with a lid formed of a parafilm. Then, the immunotube was rotated upward down in a rotator for ten minutes.
  • the solution was moved to a tube containing 1 mL of 0.5M Tris/HCl (pH: 6.8). Again, the extraction of the phase was repeated using a 100 mM trimethylamine solution (1 milliliter). In this way, 3 mL of the extraction liquid was obtained.
  • the extraction liquid (1 mL) was mixed with 9 mL of coli bacteria HST02. The mixture solution was left at rest for one hour at temperature of 30 Celsius degrees.
  • Second panning was conducted identically to the procedure of the first panning. In other words, the panning was repeated. In this way, the monoclonal phases on which the VHH antibody was displayed were purified.
  • a colony of the coli bacteria was picked up with a toothpick. The picked-up one colony was put onto one well of 96-flat-bottom plate. This was repeated. One well contained 200 microliter of a 2YTAG culture medium.
  • the solutions included in the wells were stirred at a rotation speed of 213 rpm at temperature of 30 Celsius degrees.
  • the solution (50 microliters) containing grown coli bacteria was collected.
  • the collected solution was mixed with 50 microliters of a 2YTA culture medium included in a plate.
  • the 2YTA culture medium contained helper phases such that the multiplicity of infection (i.e., MOI) was set to be 20.
  • the solution was left at rest at temperature of 37 Celsius degrees for forty minutes.
  • the plate including the 2YTA culture medium was subject to centrifugation at 1,800 rpm for twenty minutes. The supernatant was removed. The precipitate contained the coli bacteria. The precipitate was mixed with 200 microliters of a 2YTAK culture medium. The mixture solution was left at rest overnight at temperature of 30 Celsius degrees.
  • the mixture solution was subject to centrifugation at 1800 rpm for twenty minutes.
  • the supernatant containing the coli bacteria was collected.
  • intranuclear protein solution having a concentration of 2 micrograms/milliliter was injected as an antigen into each of the wells of 96-well plate (available from Thermo scientific company, trade name: maxisorp).
  • the volume of the intranuclear protein solution in each well was 50 microliters.
  • the 96-well plate was left at rest at room temperature for one hour. In this way, the NP antigen was immobilized in each well.
  • each of the wells was washed with PBS three times. Then, PBS containing 3% skim milk (available from Wako Pure Chemical Industries, Ltd.) was injected into each well (200 microliters/well). The 96-well plate was left at rest at room temperature for one hour. In this way, the intranuclear protein was blocked in each well. Subsequently, each well was washed three times with PBS.
  • a color-producting agent (available from Thermo Scientific, trade name: 1-step ultra TMB-ELISA) was injected into each well (50 microliters/well). The 96-well plate was left at rest for two minutes to cause the color-producting agent to react with the antibody.
  • a sulfuric acid aqueous solution (normal, i.e., 1N) was injected into each well at a concentration of 50 microliters/well to cease the reaction.
  • the absorbance of the solution at a wavelength of 450 nanometers was measured.
  • the protein synthesized from the DNA sequence represented by SEQ ID NO: 18 consists of the following amino acid sequence.
  • a vector pRA2(+) was purchased from Merck Millipore Company as an expression vector (See FIG. 2 ). Using In-Fusion HD Cloning Kit (available from Takara Bio Inc.), the VHH sequence was ligated into a vector pRA2(+). Hereinafter, the ligation process will be described below in more detail.
  • a VHH antibody gene fragment was amplified by the PCR method using the following two primers (SEQ ID NO: 17 and SEQ ID NO: 18) from the plasmid Vector 1 in which the VHH antibody gene fragment included in the gene library of the VHH antibody was ligated.
  • SEQ ID NO: 19 the following one DNA (SEQ ID NO: 19) including a gene sequence coding for the amino acid sequence represented by the SEQ ID NO: 08 was obtained.
  • Primer 1 (SEQ ID NO: 19) 5′ - CAGCCGGCCATGGCTGCTCAGGTGCAGCTCGTGGAGTC -3′
  • Primer 2 (SEQ ID NO: 20) 5′ - ATGGTGGCGGCCGCGTGAGGAGACGGTGACCTGGGTCC -3′
  • SEQ ID NO: 21 5′ -CAGCCGGCCATGGCTGCTCAGGTGCAGCTCGTGGAGTCTGGGGGAG GCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTCGA ACCATCTTCAATCCGAATGTCATGGGCTGGTACCGCCAGGCTCCAGGGAA GCAGCGCGAGTTGGTCGCAGATATTAGTTTAAGTGGCAGCACAAACTATG CAGACTCCGTGAAGGGCCGATTCACGATCTCCAGACAACGCCAAGAAC ACGATGTATCTGCAAATGAACAGCCTGAAGCCTGAGGATACAGCCGTCTA TTATTGTAATACTAATGCGATCAGCGGTGCGCCCG
  • a part of the base sequence included in the vector pRA2 was amplified by a PCR method using the following two primers (SEQ ID NO: 22 and SEQ ID NO: 23). In this way, a DNA (SEQ ID NO: 25) was obtained.
  • Primer 1 (SEQ ID NO: 22) 5′ - CGCGGCCGCCACCATCATCACCACCATTAATAG-3′
  • Primer 2 (SEQ ID NO: 23) 5′ - AGCCATGGCCGGCTGGGCCGCGAGTAATAAC-3′ (SEQ ID NO: 25) CGCGGCCGCCACCATCATCACCACCATTAATAGcactagtcaagaggatc cggctgctaacaaagcccgaaaggaagctgagttggctgctgcaccgct gagcaataactagcataaccccttggggcctctaaacgggtcttgagggg ttttgctgaagggg ttttgctgaaggaggaactatatccggatgaattccgtgtattctat agtgtcacctaaatcgtatgatacataaggttat
  • DNAs other than the following two DNAs (I) and (II) were fragmented with a restriction enzyme Dpnl (available from TOY0B0). In other words, the following two DNAs (I) and (II) were remained unchanged; however, the rest of the DNAs were fragmented.
  • Dpnl available from TOY0B0
  • the DNA represented by SEQ ID NO: 21 was fused with the DNA represented by the SEQ ID NO: 25. In this way, the VHH antibody gene fragment was ligated into the vector pRA2(+).
  • the ligation solution (10 microliters) and coli bacteria JM109 (available from Takara Bio, 100 microliters) were mixed on an ice.
  • the mixture solution was left at rest on the ice for thirty minutes.
  • the mixture solution was heated at temperature of 42 Celsius degrees for forty five minutes.
  • the mixture solution was left at rest on the ice for three minutes. This procedure is known as a general heat shock method.
  • the plasmids contained in the incubated coli bacteria were extracted from the LBA culture medium using a plasmid extraction kit (available from Sigma, trade name: Gene Elute Plasmid Mini Kit).
  • a plasmid extraction kit available from Sigma, trade name: Gene Elute Plasmid Mini Kit.
  • Greiner Company for the analysis of the sequence, a general T7 promotor primer set was used.
  • Coli bacteria (Competent Cell BL21 (DE3) pLysS, available from Life technologies Company) were transfected with the selected plasmids.
  • the collected coli bacteria solution (1 milliliter) was distributed onto a LBA culture medium.
  • the LBA culture medium was left at rest overnight at temperature of 37 Celsius degrees.
  • One colony was selected from among the colonies formed in the LBA culture medium.
  • the selected colony was picked up with a toothpick.
  • the picked-up colony was incubated in a LBA culture medium (3 milliliters) at temperature of 37 Celsius degrees, while shaken at 213 rpm. In this way, a culture liquid was obtained.
  • the culture liquid (3 milliliters) was mixed with a LBA culture medium (1,000 milliliters). Until the absorbance of the mixture solution at a wavelength of 600 nanometers was 0.6, the mixture solution was shaken at 120 rpm at temperature of 28 Celsius degrees.
  • IPTG solution an isopropylthiogalactoside solution
  • the final concentration of the IPTG solution was 0.5 mM.
  • the coli bacteria contained in the mixture solution were incubated at temperature of 20 Celsius degrees for overnight. In order to collect the thus-incubated coli bacteria, the mixture solution was subject to centrifugation at 6,000 rpm for ten minutes at temperature of 4 Celsius degrees.
  • the collected coli bacteria were mixed with a mixture solvent containing 50 mM Tris-HCl, 500 mM NaCl, and 5 mM imidazole.
  • the mixture solvent had a volume of 50 milliliters.
  • the coli bacteria contained in the mixture solution was disintegrated with an ultrasonic wave.
  • the disintegration liquid containing coli bacteria was subject to centrifugation at 40,000 g for thirty minutes at temperature of 4 Celsius degrees to obtain an eluate. The supernatant was collected. The collected supernatant was filtered through a 0.45-micrometer filter.
  • the filtrate was purified with Ni-NTA-Agarose (available from QIAGEN) in accordance with recommended protocol. Upon the purification, an elution buffer having a total amount of 3 microliters was used for 1 milliliter of Ni-NTA-Agarose.
  • the eluate containing the anti-NP antibody was purified with a column chromatography (available from General Electric Company, trade name: Akita purifier). In this way, a solution containing the anti-NP antibody was obtained.
  • the anti-NP antibody contained in the thus-obtained solution was quantified with an absorption spectrometer (available from Scrum Inc., trade name: nanodrop) on the basis of the absorption measurement value at a wavelength of 280 nanometers.
  • the concentration of the anti-NP antibody was 2.32 milligrams/milliliter.
  • the anti-NP antibody was evaluated as below with a recombinant NP and a surface plasmon resonance evaluation device.
  • the details of the surface plasmon resonance (hereinafter, referred to as “SPR”) will be described below.
  • the anti-Flag antibody was immobilized in accordance with the wizard included in the control software of the SPR evaluation device T200.
  • an acetic acid solution having a pH of 5.0 was used for the immobilization of the anti-Flag antibody.
  • the anti-NP antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 was used as an analyte.
  • concentrations of the anti-BP antibody contained in the running buffer were adjusted to 0.195 nM, 0.39 nM, 0.78 nM, 1.56 nM, 3.125 nM, 6.25 nM, and 12.5 nM, respectively.
  • the recombinant intranuclear proteins were captured with the anti-Flag antibodies.
  • the anti-NP antibodies were supplied. In this way, the anti-NP antibodies were evaluated.
  • FIGS. 3A-3G are graphs showing an evaluation result outputted from the SPR evaluation device T200.
  • the dissociation constant Kd was calculated using the evaluation software (available from GE Healthcare). As a result, the dissociation constant Kd was 0.236 nM.
  • VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 to recombinant nucleoproteins (namely, NP) derived from influenza subtype viruses type A H2N2, H3N2, and H7N9
  • NP recombinant nucleoproteins
  • a solution containing a recombinant nucleoprotein derived from influenza viruses subtype A H1N1 (available from Sino Biological Company, trade name: 11675-V08H) was prepared at a concentration of 500 micrograms/milliliter.
  • a solution containing a recombinant nucleoprotein (available from ORLA) derived from influenza viruses subtype B was prepared at a concentration of 500 micrograms/milliliter.
  • the six solutions are referred to as “Solution group A”.
  • a part of each of the six solutions included in the solution group A was diluted 50 fold with a PBS containing both of 5% skim milk (available from Wako Pure Chemical Industries Ltd.) and 0.05% of tween 20 (hereinafter, this PBS is referred to as “skim-milk-containing PBST”).
  • this PBS is referred to as “skim-milk-containing PBST”.
  • a diluted solution group B (Concentration: 10 micrograms/milliliter) including six diluted solutions of the recombinant NP was obtained.
  • diluted solution group B Apart of each of the six solutions included in the diluted solution group B was diluted 10-fold again with the skim-milk-containing PBST. In this way, a diluted solution group C (Concentration: 1 microgram/milliliter) including six diluted solutions of the recombinant NP was obtained.
  • a diluted solution group D (Concentration: 0.1 microgram/milliliter), a diluted solution group E (Concentration: 0.01 microgram/milliliter), a diluted solution group F (Concentration: 0.001 microgram/milliliter), a diluted solution group G (Concentration: 1 ⁇ 10 ⁇ 4 microgram/milliliter), and a diluted solution group H (Concentration: 1 ⁇ 10 ⁇ 5 microgram/milliliter).
  • the solution containing the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 was injected to the wells of 96-well microplate (Maxisorp, Nunc). Each of the wells contained 200 microliters of the solution.
  • the 96-well plate was left at rest at room temperature for two hours to immobilize the virus in the wells.
  • the skim-milk-containing PBST was injected in each well to block the virus.
  • the volume of the PBST injected to each well was 250 microliters.
  • the 96-well plate was left at rest at room temperature for three hours.
  • PBST containing 0.05% tween 20 was injected in each well to wash the wells.
  • the PBST had a pH of 7.4.
  • the volume of the PBS injected to each well was 200 microliters. This was repeated twice.
  • Each of the diluted solutions of the recombinant NP included in the diluted solution groups B-H were injected into each well.
  • the skim-milk-containing PBST was injected to another well. This well including the skim-milk-containing PBST only was used as a reference to remove a background upon measurement.
  • the volume of the solutions injected in each well was 100 microliters.
  • the 96-well plate was left at rest at room temperature. In this way, the recombinant NPs contained in the solutions B-G was bound to the anti-NP VHH antibodies contained in the wells.
  • the 96-well plate was left at rest at room temperature for one hour.
  • PBS containing 0.05% tween 20 was injected in each well to wash the wells.
  • the PBS had a pH of 7.4.
  • the volume of the PBS injected to each well was 200 microliters. This was repeated five times.
  • the anti-NP antibody (available from abcam company, trade name: ab110661) was diluted with the PBST containing 0.05% tween 20 (concentration: 5 micrograms/milliliter), and injected to each well.
  • the volume of the PBST injected to each well was 100 microliters. In this way, the anti-NP antibody was bound to the recombinant NPs contained in the wells.
  • the 96-well plate was left at rest at room temperature for one hour.
  • PBS containing 0.05% tween 20 was injected in each well to wash the wells.
  • the PBS had a pH of 7.4.
  • the volume of the PBS injected to each well was 200 microliters. This was repeated five times.
  • Labelled antibodies (available from Santa Cruz company, trade name: goat anti-mouse IgG-HRP) were diluted 20,000 fold with PBST containing 0.05% tween 20. The thus-diluted labelled antibodies was inject to each well (50 microliters/well). Then, the 96-well plate was left at rest for one hour.
  • PBS containing 0.05% tween 20 was injected in each well to wash the wells.
  • the PBS had a pH of 7.4.
  • the volume of the PBS injected to each well was 200 microliters. This was repeated five times.
  • the color-producting agent (available from Thermo Scientific, trade name: 1-step ultraTMB-ELISA) was injected into each well (50 microliters/well). The 96-well plate was left at rest for thirty minutes to cause the color-producting agent to react with the antibody.
  • the color-stopping agent (available from ScyTek laboratories, trade name: TMB Stop Buffer) containing sulfuric acid and hydrochloric acid at a low concentration was injected into each well at a concentration of 50 microliters/well to cease the reaction.
  • FIGS. 4A-4F are graphs showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type A H1N1 11675, H1N1 40205, H2N2, H3N2, H7N9, and the influenza virus type B, respectively.
  • the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 has high cross reactivity to the recombinant intranuclear proteins derived from influenza viruses type A H1N1, H2N2, H3N2, H7N9.
  • the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 has low cross reactivity to the influenza virus type B.
  • the present invention provides a composite comprising a novel antibody capable of binding to the intranuclear protein of the influenza virus.
  • the present invention also provides a detection device and a detection method using the composite comprising the novel antibody.

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Abstract

The present invention provides a composite comprising a novel antibody and at least one selected from the group consisting of a solid phase support and a labeled substance. The antibody consists of the amino acid sequence represented by SEQ ID NO: 08, and is capable of binding to an intranuclear protein of an influenza virus type A. The influenza virus type A is at least one selected from the group consisting of H1N1, H2N2, H3N2, and H7N9. The antibody is bound to the at least one selected from the group consisting of the solid phase support and the labeled substance. The present invention also provides a detection device and a detection method using the composite.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
This is a continuation-in-part of U.S. application Ser. No. 15/296,194, with a filing date of Oct. 18, 2016, which in turn claims priority of Provisional Patent Application Ser. No. 62/344,161, which was filed on Jun. 1, 2016, the contents of which Applications are hereby incorporated by reference.
INCORPORATION BY REFERENCE SEQUENCE LISTING
The material contained in the ASCII text file named “P0625743US03_ST25.txt” created on May 31, 2017, and having a file size of 20,747 bytes is incorporated by reference herein.
BACKGROUND 1. Technical Field
The present invention relates to a composite comprising an antibody capable of binding to an intranuclear protein of an influenza virus. The present invention also relates to a detection device and a detection method using the composite.
2. Description of the Related Art
Patent Literature 1 discloses antibodies each capable of binding to an influenza virus. At least a part of the antibodies disclosed in Patent Literature 1 are derived from an alpaca. Patent Literature 1 is incorporated herein by reference.
CITATION LIST Patent Literature
United States Patent Application Publication No. 2014/0302063
SUMMARY
An object of the present invention is to provide a composite comprising a novel antibody capable of binding to an intranuclear protein of an influenza virus. Another object of the present invention is to provide a detection device and a detection method using the composite comprising the novel antibody.
The present invention is a composite, comprising:
    • an antibody; and
    • at least one selected from the group consisting of a solid phase support and a labeled substance,
    • wherein
    • the antibody consists of the amino acid sequence represented by SEQ ID NO: 08, and is capable of binding to an intranuclear protein of an influenza virus type A;
    • the influenza virus type A is at least one selected from the group consisting of H1N1, H2N2, H3N2, and H7N9; and
    • the antibody is bound to the at least one selected from the group consisting of a solid phase support and the labeled substance.
The present invention provides a novel antibody capable of binding to an intranuclear protein of an influenza virus. The present invention also provides a detection device and a detection method using the composite comprising the novel antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a map of a vector used to ligate various genes included in a gene library of a VHH antibody.
FIG. 1B shows the detail of the vector map shown in FIG. 1A.
FIG. 2 is a vector map used to express the VHH antibody.
FIG. 3A is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration: 0.195 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
FIG. 3B is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration: 0.39 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
FIG. 3C is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration: 0.78 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
FIG. 3D is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration: 1.56 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
FIG. 3E is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration:3.125 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
FIG. 3F is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration: 6.25 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
FIG. 3G is a graph showing a SPR evaluation result of the binding ability of the VHH antibody (concentration: 12.5 nM) consisting of the amino acid sequence represented by SEQ ID NO: 08 to the recombinant intranuclear protein.
FIG. 4A is a graph showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type A H1N1 11675.
FIG. 4B is a graph showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type A H1N1 40205.
FIG. 4C is a graph showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type A H2N2.
FIG. 4D is a graph showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type A H3N2.
FIG. 4E is a graph showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type A H7N9.
FIG. 4F is a graph showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type B.
 DETAILED DESCRIPTION OF THE EMBODIMENT
The antibody according to the present invention is capable of binding to an influenza virus type A. In particular, the antibody according to the present invention is capable of binding to an intranuclear protein of an influenza virus type A. As disclosed in Patent Literature 1, an antibody capable of binding to an influenza virus consists of, in an N- to C-direction, the following structural domains.
N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C
where
FR denotes a framework region amino acid sequence and CDR denotes a complementary determining region amino acid sequence.
In the present invention, the CDR1 consists of an amino acid sequence represented by RTIFNPNVMG (SEQ ID NO: 01).
In the present invention, the CDR2 consists of an amino acid sequence represented by DISLSGSTNYADSVKG (SEQ ID NO: 02).
In the present invention, the CDR3 consists of an amino acid sequence represented by NAISGAPGRY (SEQ ID NO: 03).
Desirably, the CDR1, the CDR2, and the CDR3 are represented by SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03 respectively. In this case, more desirably, the FR1, the FR2, the FR3, and the FR4 consist of amino acid sequences represented by QVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 04), WYRQAPGKQRELVA (SEQ ID NO: 05), RFTISRDNAKNTMYLQMNSLKPEDTAVYYCNT (SEQ ID NO: 06), and WGQGAQVTVSS (SEQ ID NO: 07), respectively. In other words, it is desirable that the antibody according to the present invention consists of the following amino acid sequence.
(SEQ ID NO: 08)
QVQLVESGGGLVQAGGSLRLSCAASRTIFNPNVMGWYRQAPGKQRELVAD
ISLSGSTNYADSVKGRFTI SRDNAKNTMYLQMNSLKPEDTAVYYCNTNA
ISGAPGRYWGQGTQVIVSS
The antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 does not exhibit antigen cross reactivity with regard to an influenza virus, such as an influenza virus type B, other than the influenza virus type A.
The antibody according to the present invention is employed in a detection device or in a detection method for detecting the intranuclear protein of an influenza virus type A. In this case, the antibody according to the present invention is used in a state where the antibody is bound to at least one selected from the group consisting of a solid support and a labeled substance.
As long as the solid support is a support insoluble in a solvent used for a reaction system of an antigen-antibody reaction, a shape and a material of the solid support is not limited. An example of the shape of the solid support is a plate, a bead, a disk, a tube, a filter, and a film. An example of a material of the solid support is a polymer such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, or polymethylmethacrylate, a metal such as gold, silver, or aluminum, or glass. A known method such as a physical adsorption method, a covalent binding method, an ion bonding method, or a cross-linking method is employed as a method for binding the antibody to the solid support.
For example, a labeled substance such as a fluorescent substance, a luminescent substance, a dye, and a radioactive substance is used. A known method such as a physical adsorption method, a covalent binding method, an ion bonding method, or a cross-linking method is employed as a method for binding the antibody to the labeled substance.
In the detection method in which the antibody according to the present invention is used, the composite including the antibody is brought into contact with an analyte. Then, detected is a change of a physical amount based on an antigen-antibody reaction of the intranuclear protein of the influenza virus type A contained in the analyte and the antibody included in the composite. An example of the physical amount is, for example, luminescence intensity, chromaticity, light transmission, turbidness, absorbance, or radiation dose. A known method such as an enzyme immunoassay method, an immunochromatography method, a latex agglutination method, a radioimmunoassay method, a fluorescence immunoassay method, or a surface plasmon resonance spectroscopy method is employed as an example of the detection method.
The detection device in which the antibody according to the present invention is employed includes a detector for detecting any one of the physical amount which may be changed on the basis of the antigen-antibody reaction. The detector is composed of a known device such as a photometer, a spectroscope, or a dosimeter.
EXAMPLES Inventive Example 1
VHH antibodies capable of binding to an intranuclear protein included in an influenza virus type A H1N1 were prepared in accordance with the following procedures. Hereinafter, the intranuclear protein may be referred to as “NP”.
(Immunization of Alpaca and Acquirement of Mononuclear)
In order to form a VHH antibody gene library, an alpaca was immunized using a recombinant intranuclear protein (SEQ ID NO: 24) derived from an Influenza virus type A H1N1 (A/Puerto Rico/8/34/Mount Sinai) as an antigen. The recombinant intranuclear protein was prepared using a Brevibacillus expression system by Higeta Shoyu Co., Ltd. The recombinant intranuclear protein was adjusted with an adjuvant before administrated to an alpaca.
The recombinant intranuclear protein used in the inventive example 1 is shown below.
(SEQ ID NO: 24)
MASQGTKRSYEQMETDGERQNATEIRASVGKMIGGIGRFYIQMCTELKLS
DYEGRLIQNSLTIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRV
NGKWMRELILYDKEEIRRIWRQANNGDDATAGLTHMMIWHSNLNDATYQR
TRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELVRMIKRG
INDRNFWRGENGRKTRIAYERMCNILKGKFQTAAQKAMMDQVRESRNPGN
AEFEDLTFLARSALILRGSVAHKSCLPACVYGPAVASGYDFEREGYSLVG
IDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVLSFIKGT
KVLPRGKLSTRGVQIASNENMETMESSTLELRSRYWAIRTRSGGNTNQQR
ASAGQISIQPTFSVQRNLPFDRTTIMAAFNGNTEGRTSDMRTEIIRMMES
ARPEDVSFQGRGVFELSDEKAASPIVPSFDMSNEGSYFFGDNAEEYDN
Specifically, the recombinant intranuclear protein having a concentration of 100 micrograms/milliliter was administered to the alpaca. After one week, the recombinant HA protein having the same concentration was administered to the alpaca, again. In this way, the alpaca was immunized with the recombinant HA protein five times for five weeks. After another week, blood of the alpaca was extracted. Then, mononuclear cells were acquired from the blood as below.
A blood cell separation solution (available from COSMO BIO Co., Ltd., trade name: Lymphoprep) was added to a lymphocyte separation tube (available from Greiner Bio-One Co., Ltd., trade name: Leucosep). Then, the solution was subject to centrifugation under a temperature of 20 Celsius degrees at 1,000×g for one minute.
The blood extracted from the alpaca was treated with heparin. Then, an equivalent amount of phosphate buffered saline (hereinafter, referred to as “PBS”) was added to the thus-treated blood to obtain a sample solution. Then, the sample solution was added to the lymphocyte separation tube to which the blood cell separation solution was added.
The lymphocyte separation tube was subject to centrifugation under a temperature of 20 Celsius degrees at 800×g for thirty minutes.
The fraction containing the mononuclear cells was collected. PBS having three times volume was added. The fraction was subject to centrifugation under a temperature of 20 Celsius degrees at 300×g for five minutes. The precipitate was suspended with PBS mildly. After the suspending, 10 microliters of the suspension was separated in order for the count of the number of the cell. The remaining suspension was subject to centrifugation under a temperature of 20 Celsius degrees at 300×g for five minutes.
A RNA storage solution (trade name: RNAlater) having a volume of 2 milliliters was added to the precipitate. Then, the solution was suspended mildly. The suspension was injected to two tubes each having a volume of 1.5 milliliters. Each tube included 1 milliliter of the suspension. The tube was stored under a temperature of −20 Celsius degrees. The suspension (5 microliters) separated for the count of the number of the cell was mixed with a Tork's solution (15 microliters), and the number of the mononuclear cells was counted with a counting chamber.
(Formation of cDNA Gene Library of VHH Antibody)
Then, a total RNA was extracted from the mononuclear cells, and a cDNA gene library of VHH antibody was formed in accordance with the following procedure. In the following procedure, RNase-free-grade reagents and instruments were used.
A total RNA isolation reagent (Trade name: TRIzol Regent, 1 milliliter) was added to the mononuclear cell fraction. The reagent was mixed mildly with the fraction, and left at rest at room temperature for five minutes. Chloroform (200 microliters) was added to the reagent, and the reagent was shaken strongly for fifteen seconds. The reagent was left at a room temperature for two-three minutes. The reagent was subject to centrifugation under a temperature of 4 Celsius degrees for 15 minutes at 12,000 or less x g.
The supernatant was moved to a new tube. RNase-free water and chloroform (200 microliters, each) were added to the tube. In addition, 500 milliliters of isopropanol was added to the tube. The liquid included in the tube was stirred with a vortex mixer. The liquid was left at rest at room temperature for ten minutes. Then, the liquid was subject to centrifugation under a temperature of 4 Celsius degrees for fifteen minutes at 12,000 or less x g. The supernatant was removed, and the precipitate was rinse with one milliliter of 75% ethanol. This solution was subject to centrifugation under a temperature of four Celsius degrees for five minutes at 7,500 or less x g. The solution was dried to obtain total RNA. The obtained total RNA was solved in RNase-free water.
In order to obtain cDNA from the total RNA, a kit including a reverse transcriptase was employed. The kit was available from Takara Bio Inc., as a trade name of PrimeScript II 1st strand cDNA Synthesis Kit. The Random 6 mer and Oligo dT primer included in the kit were used as primers. The cDNA was obtained in accordance with the standard protocol attached in the kit.
The gene of the VHH antibody included in the alpaca was obtained from the cDNA by a PCR method. An enzyme for PCR was available from Takara Bio Inc., as a trade name of Ex-taq.
The following reagents were mixed to obtain a mixture solution.
10x buffer 5 microliters
dNTPs
4 microliters
Primer F
2 microliters
Primer R
2 microliters
cDNA template
1 microliter
Ex-taq 0.25 microliters
The mixture solution was subject to the following PCR method.
First, the mixture solution was heated at temperature of 95 Celsius degrees for two minutes.
Then, the temperature of the mixture solution was varied in accordance with the following cycle.
    • Ninety six Celsius degrees for thirty seconds,
    • Fifty two Celsius degrees for thirty seconds, and
    • Sixty eight Celsius degrees for forty seconds
This cycle was repeated thirty times.
Finally, the mixture solution was heated at temperature of sixty eight Celsius degrees for four minutes and stored at temperature of four Celsius degrees.
The following primers were used in the present PCR method.
(SEQ ID NO: 09)
Primer 1: 5′ - GGTGGTCCTGGCTGC -3′
(SEQ ID NO: 10)
Primer 2: 5′ - ctgctcctcgcGGCCCAGCCGGCCatggcTSAGKT
GCAGCTCGTGGAGTC -3′
(SEQ ID NO: 11)
Primer 3: 5′ - TGGGGTCTTCGCTGTGGTGCG -3′
(SEQ ID NO: 12)
Primer 4: 5′ - TTGTGGTTTTGGTGTCTTGGG -3′
(SEQ ID NO: 13)
Primer 5: 5′ - tttgCtctGCGGCCGCagaGGCCgTGGGGTCTTCG
CTGTGGTGCG -3′
(SEQ ID NO: 14)
Primer 6: 5′ -tttgCtctGCGGCCGCagaGGCCgaTTGTGGTTTTG
GTGTCTTGGG-3′
(Reference literature: Biomed Environ Sci., 2012; 27(2): 118-121)
Three PCR assays were conducted.
In the first PCR assay, a primer set A composed of the cDNA, Primer 1 and Primer 3 and a primer set B composed of the cDNA, Primer 1 and Primer 4 were used.
In the second PCR assay, a primer set C composed of the gene amplified with the primer set A, Primer 2, and Primer 3, and a primer set D composed of the gene amplified with the primer set B, Primer 2, and Primer 4.
In the third PCR assay, a primer set E composed of the gene amplified with the primer set C, Primer 2, and Primer 5, and a primer set F composed of the gene amplified with the primer set D, Primer 2, and Primer 6.
In this way, the gene library of the VHH antibody was formed. In other words, the gene library of the VHH antibody included the genes amplified with the primer sets E and F.
(Formation of Phage Library)
Next, a phase I library was formed from the gene library of the VHH antibody in accordance of the following procedures.
A plasmid Vector 1 (4057 bp, see FIG. 1A) derived from a commercially available plasmid pUC119 (for example, available from Takara Bio. Inc.,) was treated with a restriction enzyme SfiI. The restriction enzyme site SfiI(a) consists of the gene sequence represented by GGCCCAGCCGGCC (SEQ ID NO: 15). The restriction enzyme site SfiI(b) consists of the gene sequence represented by GGCCTCTGCGGCC (SEQ ID NO: 16). FIG. 1B shows a detail vector map of the plasmid Vector 1.
The plasmid Vector 1 consists of the following gene sequence.
(SEQ ID NO: 17)
gacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgata
ataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcgg
aacccctatttgtttatttttctaaatacattcaaatatgtatccgctca
tgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagt
atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcatt
ttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatg
ctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaac
agcggtaagatccttgagagttttcgccccgaagaacgttttccaatgat
gagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacg
ccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttg
gttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagt
aagagaattatgcagtgctgccataaccatgagtgataacactgcggcca
acttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttg
cacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagct
gaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaa
tggcaacaacgttgcgcaaactattaactggcgaactacttactctagct
tcccggcaacaattaatagactggatggaggcggataaagttgcaggacc
acttctgcgctcggcccttccggctggctggtttattgctgataaatctg
gagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagat
ggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaac
tatggatgaacgaaatagacagatcgctgagataggtgcctcactgatta
agcattggtaactgtcagaccaagtttactcatatatactttagattgat
ttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttga
taatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgt
cagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctg
cgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggt
ttgtttgccggatcaagagctaccaactctttttccgaaggtaactggct
tcagcagagcgcagataccaaatactgtccttctagtgtagccgtagtta
ggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgct
aatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccg
ggttggactcaagacgatagttaccggataaggcgcagcggtcgggctga
acggggggttcgtgcacacagcccagcttggagcgaacgacctacaccga
actgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaag
ggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagag
cgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgt
cgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcag
gggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttc
ctggccttttgctggccttttgctcacatgttctttcctgcgttatcccc
tgattctgtggataaccgtattaccgcctttgagtgagctgataccgctc
gccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaa
gagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcatta
atgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgca
acgcaattaatgtgagttagctcactcattaggcaccccaggctttacac
tttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatt
tcacacaggaaacagctatgaccatgattacgccAAGCTTCGAAGGAGAC
AGTCATAatgaaatacctgctgccgaccgctgctgctggtctgctgctcc
tcgcGGCCCAGCCGGCCatggagcTCAAGATGACACAGACTACATCCTCC
CTGTCAGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCA
GGACATTAGCGATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTG
TTAAACTCCTGATCTATTACACATCAAGTTTACACTCAGGAGTCCCATCA
AGGTTCAGTGGCGGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAA
CCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGC
TTCCGTGGACGTTTGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGAT
GCTGCACCAACTgtaGGCCtctGCGGCCGCagaGcaaaaactcatctcag
aagaggatctgaatggggccgcaTAGggttccggtgattttgattatgaa
aagatggcaaacgctaataagggggctatgaccgaaaatgccgatgaaaa
cgcgctacagtctgacgctaaaggcaaacttgattctgtcgctactgatt
acggtgctgctatcgatggtttcattggtgacgtttccggccttgctaat
ggtaatggtgctactggtgattttgctggctctaattcccaaatggctca
agtcggtgacggtgataattcacctttaatgaataatttccgtcaatatt
taccttccctccctcaatcggttgaatgtcgcccttttgtctttagcgct
ggtaaaccatatgaattttctattgattgtgacaaaataaacttattccg
tggtgtctttgcgtttcttttatatgttgccacctttatgtatgtatttt
ctacgtttgctaacatactgcgtaataaggagtctTAATAAgaattcact
ggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaac
ttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaa
gaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcga
atggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcac
accgCATATGaAAATTGTAAgcgttaatattttgttaaaattcgcgttaa
atttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaa
atcccttataaatcaaaagaatagaccgagatagggttgagtgttgttcc
agtttggaacaagagtccactattaaagaacgtggactccaacgtcaaag
ggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccc
taatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccc
taaagggagcccccgatttagagcttgacggggaaagccggcgaacgtgg
cgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggca
agtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgc
gccgctacaGGGCGCGTcccatATGgtgcactctcagtacaatctgctct
gatgccgcatagttaagccagccccgacacccgccaacacccgctgacgc
gccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtga
ccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaa
cgcgcga
Similarly, the gene library of the VHH antibody was treated with the restriction enzyme SfiI. In this way, VHH antibody gene fragments were obtained.
The thus-treated plasmid Vector 1 was mixed with the VHH antibody gene fragments at a ratio of 1:2. An enzyme (available from Toyobo Co. Ltd., trade name: Ligation High ver. 2) was injected to the mixture solution. The mixture solution was left at rest at a temperature of 16 Celsius degrees for two hours. In this way, each of the VHH anti body gene fragments was ligated into the plasmid Vector 1.
Coli bacteria (available from Takara Bio. Inc., trade name: HST02) were transfected with the thus-ligated plasmid Vector 1.
Then, the coli bacteria were incubated for fifteen hours on a 2YT plate culture medium containing ampicillin at a concentration of 100 micrograms/milliliter. In this way, obtained was a library of phases each of which displays a protein obtained from the gene fragment included in the gene library of the VHH antibody.
After the incubation, a concentration of the library was calculated by counting the number of single colonies formed on the 2YT plate culture medium. As a result, the library of the phases had a concentration of 5×107/milliliter.
(Biopanning)
VHH antibodies capable of specifically binding to the intranuclear protein were obtained from the phage library in accordance with the following procedures.
In order to extract the clones each capable of the antigen from among the phases which expressed the VHH antibody, biopanning was conducted twice.
Coli bacteria (HST02) to which the VHH antibody gene fragment included in the gene library of the VHH antibody was introduced were incubated at temperature of 30 Celsius degrees in the 2YT AG culture medium containing 100 micrograms/milliliter of ampicillin and 1% glucose in such a manner that a value OD600 indicating absorbance reached 1.0. The 2YT AG culture has a volume of 100 milliliters. In this way, the Coli bacteria were proliferated.
Helper phages (available from Invitrogen company, trade name: M13K07) were added to the coli bacteria culture medium in such a manner that the multiplicity of infection was approximately twenty.
Then, the culture medium was warmed for about thirty minutes at temperature of 37 Celsius degrees. Then, the culture medium was subject to centrifugation at rotation speed of 4000 rpm for ten minutes to collect the coli bacteria. The coli bacteria was incubated overnight at temperature of 30 Celsius degrees in a 2YTAK culture medium containing 100 micrograms/milliliter of ampicillin and 1% 50 micrograms/milliliter of kanamycin, while subjected to centrifugation at 213 rpm.
The incubation liquid (100 milliliters) containing the thus-incubated coli bacteria were injected to two centrifugation tubes(volume: 50 milliliters, each). The two centrifugation tubes were subject to centrifugation for ten minutes at rotation speed of 4,000 rpm. Then, the supernatants (20 milliliters, each) were collected.
The supernatants (40 milliliters) were added to a 20% polyethylene glycol solution (10 milliliters) containing NaCl (2.5M). Then, the mixture solution was mixed upward down. Subsequently, the mixture solution was cooled on an ice for approximately one hour. The mixture was subject to centrifugation for ten minutes at rotation speed of 4,000 rpm. Then, the supernatant was removed. PBS containing 10% glycerol was injected toward the precipitate. Finally, the precipitate was loosened and dissolved. In this way, a library of phases each of which displays the VHH antibody was obtained.
(Screening of the VHH Antibody Capable of Specifically Binding to NP)
(A) Immobilization of NP Antigen
NP was mixed with PBS to prepare an NP solution. The concentration of NP was 2 micrograms/milliliter. The NP solution (2 milliliters) was injected into an immunotube (available from NUNC Co. Ltd.). The NP solution was left at rest in the immunotube for one hour. In this way, NP was immobilized in the immunotube.
Then, the inside of the immunotube was washed three times with PBS.
The inside of the immunotube was filled with PBS which contained 3% skim milk (available from Wako Pure Chemical Industries, Ltd.). In this way, NP was blocked as an antigen in the immunotube.
The immunotube was left at rest at room temperature for one hour. Subsequently, the inside of the immunotube was washed three times with PBS.
(B) Panning
The library of the phages each of which displays the VHH antibody (Concentration: approximately 10E+11/milliliter) was mixed with 3 milliliters of PBS containing 3% skim milk to prepare a mixture solution. The mixture solution was injected into the immunotube in which the NP antigen was immobilized.
The immunotube was provided with a lid formed of a parafilm. Then, the immunotube was rotated upward down in a rotator for ten minutes.
The immunotube was left at rest at room temperature for one hour.
The inside of the immunotube was washed ten times with PBS containing 0.05% tween 20. Hereinafter, such a PBS is referred to as “PBST”.
The inside of the immunotube was filled with PBST. Subsequently, the immunotube was left at rest for ten minutes. Then, the inside of the immunotube was washed ten times with PBST.
In order to extract phases each of which displays the VHH antibody bound to the NP antigen, 100 mM trimethylamine solution (1 milliliters) was injected to the immunotube.
The immunotube was provided with a lid formed of a parafilm. Then, the immunotube was rotated upward down in a rotator for ten minutes.
In order to neutralize the solution, the solution was moved to a tube containing 1 mL of 0.5M Tris/HCl (pH: 6.8). Again, the extraction of the phase was repeated using a 100 mM trimethylamine solution (1 milliliter). In this way, 3 mL of the extraction liquid was obtained.
The extraction liquid (1 mL) was mixed with 9 mL of coli bacteria HST02. The mixture solution was left at rest for one hour at temperature of 30 Celsius degrees.
In order to count the number of colonies, 10 microliters of the mixture solution containing the coli bacteria HST02 was distributed onto a small plate including a 2TYA culture medium (10 milliliters/plate).
The rest of the mixture solution was subject to centrifugation. The supernatant was removed, and the precipitate was distributed onto a large plate including a 2TYA culture medium (40 milliliters/plate). These two plates were left at rest overnight at temperature of 30 Celsius degrees. In this way, first panning was conducted.
Second panning was conducted identically to the procedure of the first panning. In other words, the panning was repeated. In this way, the monoclonal phases on which the VHH antibody was displayed were purified.
After the second panning, a colony of the coli bacteria was picked up with a toothpick. The picked-up one colony was put onto one well of 96-flat-bottom plate. This was repeated. One well contained 200 microliter of a 2YTAG culture medium.
The solutions included in the wells were stirred at a rotation speed of 213 rpm at temperature of 30 Celsius degrees.
The solution (50 microliters) containing grown coli bacteria was collected. The collected solution was mixed with 50 microliters of a 2YTA culture medium included in a plate. The 2YTA culture medium contained helper phases such that the multiplicity of infection (i.e., MOI) was set to be 20. The solution was left at rest at temperature of 37 Celsius degrees for forty minutes.
The plate including the 2YTA culture medium was subject to centrifugation at 1,800 rpm for twenty minutes. The supernatant was removed. The precipitate contained the coli bacteria. The precipitate was mixed with 200 microliters of a 2YTAK culture medium. The mixture solution was left at rest overnight at temperature of 30 Celsius degrees.
The mixture solution was subject to centrifugation at 1800 rpm for twenty minutes. The supernatant containing the coli bacteria was collected.
(C) Qualitative Evaluation of Phase-Displayed VHH Antibody and Antigen by ELISA
An intranuclear protein solution having a concentration of 2 micrograms/milliliter was injected as an antigen into each of the wells of 96-well plate (available from Thermo scientific company, trade name: maxisorp). The volume of the intranuclear protein solution in each well was 50 microliters. The 96-well plate was left at rest at room temperature for one hour. In this way, the NP antigen was immobilized in each well.
Each of the wells was washed with PBS three times. Then, PBS containing 3% skim milk (available from Wako Pure Chemical Industries, Ltd.) was injected into each well (200 microliters/well). The 96-well plate was left at rest at room temperature for one hour. In this way, the intranuclear protein was blocked in each well. Subsequently, each well was washed three times with PBS.
(The monoclonal phases each of which displays the VHH antibody were injected into each well (50 microliters/well). Then, the 96-well plate was left at rest for one hour. In this way, the phases reacted with the NP antigen.
Each well was washed three times with PBST. Then, anti-M13 antibody (available from ABCAM company, trade name; ab50370, 10,000-fold dilution) was injected into each well (50 microliters/well). Then, each well was washed three times with PBST.
A color-producting agent (available from Thermo Scientific, trade name: 1-step ultra TMB-ELISA) was injected into each well (50 microliters/well). The 96-well plate was left at rest for two minutes to cause the color-producting agent to react with the antibody.
A sulfuric acid aqueous solution (normal, i.e., 1N) was injected into each well at a concentration of 50 microliters/well to cease the reaction.
The absorbance of the solution at a wavelength of 450 nanometers was measured.
Six wells each having good absorbance measurement result were selected. The DNA sequences included in the phases contained in the selected six wells were analyzed by Greiner Company. The analysis results of the DNA sequences will be described below. The following one DNA sequences were found.
(SEQ ID NO: 18)
GCTCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGG
GTCTCTGAGACTCTCCTGTGCAGCCTCTCGAACCATCTTCAATCCGAATG
TCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCA
GATATTAGTTTAAGTGGCAGCACAAACTATGCAGACTCCGTGAAGGGCCG
ATTCACGATCTCCAGAGACAACGCCAAGAACACGATGTATCTGCAAATGA
ACAGCCTGAAGCCTGAGGATACAGCCGTCTATTATTGTAATACTAATGCG
ATCAGCGGTGCGCCCGGAAGGTACTGGGGCCAGGGGACCCAGGTCACCGT
CTCCTCA
The protein synthesized from the DNA sequence represented by SEQ ID NO: 18 consists of the following amino acid sequence.
(SEQ ID NO: 08)
QVQLVESGGGLVQAGGSLRLSCAASRTIFNPNVMGWYRQAPGKQRELVAD
ISLSGSTNYADSVKGRFTI SRDNAKNTMYLQMNSLKPEDTAVYYCNTNA
ISGAPGRYWGQGTQVTVSS
(Expression of Anti-NP VHH Antibody)
A vector pRA2(+) was purchased from Merck Millipore Company as an expression vector (See FIG. 2). Using In-Fusion HD Cloning Kit (available from Takara Bio Inc.), the VHH sequence was ligated into a vector pRA2(+). Hereinafter, the ligation process will be described below in more detail.
First, a VHH antibody gene fragment was amplified by the PCR method using the following two primers (SEQ ID NO: 17 and SEQ ID NO: 18) from the plasmid Vector 1 in which the VHH antibody gene fragment included in the gene library of the VHH antibody was ligated. In this way, the following one DNA (SEQ ID NO: 19) including a gene sequence coding for the amino acid sequence represented by the SEQ ID NO: 08 was obtained.
Primer 1:
(SEQ ID NO: 19)
5′ - CAGCCGGCCATGGCTGCTCAGGTGCAGCTCGTGGAGTC -3′
Primer 2:
(SEQ ID NO: 20)
5′ - ATGGTGGCGGCCGCGTGAGGAGACGGTGACCTGGGTCC -3′
(SEQ ID NO: 21)
5′ -CAGCCGGCCATGGCTGCTCAGGTGCAGCTCGTGGAGTCTGGGGGAG
GCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTCGA
ACCATCTTCAATCCGAATGTCATGGGCTGGTACCGCCAGGCTCCAGGGAA
GCAGCGCGAGTTGGTCGCAGATATTAGTTTAAGTGGCAGCACAAACTATG
CAGACTCCGTGAAGGGCCGATTCACGATCTCCAGAGACAACGCCAAGAAC
ACGATGTATCTGCAAATGAACAGCCTGAAGCCTGAGGATACAGCCGTCTA
TTATTGTAATACTAATGCGATCAGCGGTGCGCCCGGAAGGTACTGGGGCC
AGGGGACCCAGGTCACCGTCTCCTCACGCGGCCGCCACCAT-3′
On the other hand, a part of the base sequence included in the vector pRA2 was amplified by a PCR method using the following two primers (SEQ ID NO: 22 and SEQ ID NO: 23). In this way, a DNA (SEQ ID NO: 25) was obtained.
Primer 1:
(SEQ ID NO: 22)
5′ - CGCGGCCGCCACCATCATCACCACCATTAATAG-3′
Primer 2:
(SEQ ID NO: 23)
5′ - AGCCATGGCCGGCTGGGCCGCGAGTAATAAC-3′
(SEQ ID NO: 25)
CGCGGCCGCCACCATCATCACCACCATTAATAGcactagtcaagaggatc
cggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgct
gagcaataactagcataaccccttggggcctctaaacgggtcttgagggg
ttttttgctgaaaggaggaactatatccggatgaattccgtgtattctat
agtgtcacctaaatcgtatgtgtatgatacataaggttatgtattaattg
tagccgcgttctaacgacaatatgtacaagcctaattgtgtagcatctgg
cttactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcg
gtttggttggacgaaccttctgagtttctggtaacgccgtcccgcacccg
gaaatggtcagcgaaccaatcagcagggtcatcgctagccagatcctcta
cgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctg
gcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttc
gggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggc
cgggggactgttgggcgccatctccttgcatgcaccattccttgcggcgg
cggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggag
tcgcataagggagagcgtcgaatggtgcactctcagtacaatctgctctg
atgccgcatagttaagccagccccgacacccgccaacacccgctgacgcg
ccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgac
cgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaac
gcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtc
atgataataatggtttcttagacgtcaggtggcacttttcggggaaatgt
gcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatc
cgctcatgagacaataaccctgataaatgcttcaataatattgaaaaagg
aagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgc
ggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaa
aagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggat
ctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttcc
aatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgta
ttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaat
gacttggttgagtactcaccagtcacagaaaagcatcttacggatggcat
gacagtaagagaattatgcagtgctgccataaccatgagtgataacactg
cggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgct
tttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaacc
ggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctg
tagcaatggcaacaacgttgcgcaaactattaactggcgaactacttact
ctagcttcccggcaacaattaatagactggatggaggcggataaagttgc
aggaccacttctgcgctcggcccttccggctggctggtttattgctgata
aatctggagccggtgagcgtgggtctcgcggtatcattgcagcactgggg
ccagatggtaagccctcccgtatcgtagttatctacacgacggggagtca
ggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcac
tgattaagcattggtaactgtcagaccaagtttactcatatatactttag
attgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcct
ttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccact
gagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttt
tttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagc
ggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaa
ctggcttcagcagagcgcagataccaaatactgttcttctagtgtagccg
tagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgc
tctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtc
ttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcg
ggctgaacggggggttcgtgcacacagcccagcttggagcgaacgaccta
caccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttc
ccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaaca
ggagagcgcacgagggagcttccagggggaaacgcctggtatctttatag
tcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgct
cgtcaggggggcggagcctatggaaaaacgccagcaacgcggccttttta
cggttcctggccttttgctggccttttgctcacatgttctttcctgcgtt
atcccctgattctgtggataaccgtattaccgcctttgagtgagctgata
ccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaa
gcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgat
tcattaatgcagctggcttatcgaaattaatacgactcactatagggaga
cccaagctttatttcaaggagacagtcataATGaaatacctattgcctac
ggcagccgctggattgttattactcgcggcccagccggccatggct
DNAs other than the following two DNAs (I) and (II) were fragmented with a restriction enzyme Dpnl (available from TOY0B0). In other words, the following two DNAs (I) and (II) were remained unchanged; however, the rest of the DNAs were fragmented.
    • (I) the DNA represented by SEQ ID NO: 21, and
    • (II) the DNA represented by SEQ ID NO: 25.
The DNA represented by SEQ ID NO: 21 was fused with the DNA represented by the SEQ ID NO: 25. In this way, the VHH antibody gene fragment was ligated into the vector pRA2(+).
The ligation solution (10 microliters) and coli bacteria JM109 (available from Takara Bio, 100 microliters) were mixed on an ice. The mixture solution was left at rest on the ice for thirty minutes. Then, the mixture solution was heated at temperature of 42 Celsius degrees for forty five minutes. Finally, the mixture solution was left at rest on the ice for three minutes. This procedure is known as a general heat shock method.
After the incubation at a temperature of 37 degrees Celsius for one hour with shaking, all the amount of the mixture solution was distributed onto a LBA culture medium containing ampicillin at a concentration of 100 micrograms/milliliter. The LBA culture medium was left at rest overnight at temperature of 37 Celsius degrees.
Three colonies were selected from among the colonies formed on the LBA culture medium. The selected three colonies were incubated in the LBA culture medium (3 milliliters).
The plasmids contained in the incubated coli bacteria were extracted from the LBA culture medium using a plasmid extraction kit (available from Sigma, trade name: Gene Elute Plasmid Mini Kit). In order to confirm that the gene of the targeted VHH antibody was inserted in the plasmid, the sequence of the plasmid was analyzed by Greiner Company. For the analysis of the sequence, a general T7 promotor primer set was used.
Selected were plasmids which were confirmed through the analysis of the sequence to be formed as planned.
Coli bacteria (Competent Cell BL21 (DE3) pLysS, available from Life technologies Company) were transfected with the selected plasmids.
An LBA culture medium (1 microliter) was injected to the solution containing the transfected coli bacteria. Then, the coli bacteria were rescued at temperature of 37 Celsius degrees for one hour, while shaken at 213 rpm.
Then, the coli bacteria solution was collected. The collected coli bacteria solution (1 milliliter) was distributed onto a LBA culture medium. The LBA culture medium was left at rest overnight at temperature of 37 Celsius degrees.
One colony was selected from among the colonies formed in the LBA culture medium. The selected colony was picked up with a toothpick. The picked-up colony was incubated in a LBA culture medium (3 milliliters) at temperature of 37 Celsius degrees, while shaken at 213 rpm. In this way, a culture liquid was obtained.
In addition, the culture liquid (3 milliliters) was mixed with a LBA culture medium (1,000 milliliters). Until the absorbance of the mixture solution at a wavelength of 600 nanometers was 0.6, the mixture solution was shaken at 120 rpm at temperature of 28 Celsius degrees.
After the absorbance was 0.6, an isopropylthiogalactoside solution (hereinafter, referred to as “IPTG solution”) was added to the mixture solution. The final concentration of the IPTG solution was 0.5 mM. The coli bacteria contained in the mixture solution were incubated at temperature of 20 Celsius degrees for overnight. In order to collect the thus-incubated coli bacteria, the mixture solution was subject to centrifugation at 6,000 rpm for ten minutes at temperature of 4 Celsius degrees.
The collected coli bacteria were mixed with a mixture solvent containing 50 mM Tris-HCl, 500 mM NaCl, and 5 mM imidazole. The mixture solvent had a volume of 50 milliliters. The coli bacteria contained in the mixture solution was disintegrated with an ultrasonic wave.
The disintegration liquid containing coli bacteria was subject to centrifugation at 40,000 g for thirty minutes at temperature of 4 Celsius degrees to obtain an eluate. The supernatant was collected. The collected supernatant was filtered through a 0.45-micrometer filter.
The filtrate was purified with Ni-NTA-Agarose (available from QIAGEN) in accordance with recommended protocol. Upon the purification, an elution buffer having a total amount of 3 microliters was used for 1 milliliter of Ni-NTA-Agarose.
Furthermore, the eluate containing the anti-NP antibody was purified with a column chromatography (available from General Electric Company, trade name: Akita purifier). In this way, a solution containing the anti-NP antibody was obtained.
The anti-NP antibody contained in the thus-obtained solution was quantified with an absorption spectrometer (available from Scrum Inc., trade name: nanodrop) on the basis of the absorption measurement value at a wavelength of 280 nanometers. As a result, the concentration of the anti-NP antibody was 2.32 milligrams/milliliter.
(D-1) Surface Plasmon Resonance Evaluation of Anti-NP Antibody Using Recombinant NP
The anti-NP antibody was evaluated as below with a recombinant NP and a surface plasmon resonance evaluation device. The details of the surface plasmon resonance (hereinafter, referred to as “SPR”) will be described below.
    • SPR evaluation device: T200 (available from GE Healthcare)
    • Immobilization buffer: PBS containing 0.05% of Tween 20
    • Running buffer: PBS containing 0.05% of Tween 20
    • Sensor chip: CM5 (available from GE Healthcare)
    • Immobilization reagents: N-Hydroxysuccinimide (NHS) and N-[3-(Dimethylamino)propyl]-N′-ethylcarbodiimide (EDC)
    • Anti-Flag antibody: Monoclonal ANTI-FLAG antibody (available from SIGMA)
    • NP: recombinant nucleoprotein (NP) protein derived from influenza virus H1N1 to which a Flag tag was fused and which was prepared using baculovirus
The anti-Flag antibody was immobilized in accordance with the wizard included in the control software of the SPR evaluation device T200. For the immobilization of the anti-Flag antibody, an acetic acid solution having a pH of 5.0 was used.
The anti-NP antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 was used as an analyte. In the first to seven analyses, the concentrations of the anti-BP antibody contained in the running buffer were adjusted to 0.195 nM, 0.39 nM, 0.78 nM, 1.56 nM, 3.125 nM, 6.25 nM, and 12.5 nM, respectively. First, the recombinant intranuclear proteins were captured with the anti-Flag antibodies. Then, the anti-NP antibodies were supplied. In this way, the anti-NP antibodies were evaluated. FIGS. 3A-3G are graphs showing an evaluation result outputted from the SPR evaluation device T200. The dissociation constant Kd was calculated using the evaluation software (available from GE Healthcare). As a result, the dissociation constant Kd was 0.236 nM.
(D-2) Evaluation of Cross Reactivity to Other Influenza Virus Subtype by ELISA
Next, in order to evaluate binding ability of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 to recombinant nucleoproteins (namely, NP) derived from influenza subtype viruses type A H2N2, H3N2, and H7N9, the binding ability to the recombinant intranuclear proteins was evaluated with an ELSA measurement method.
A solution containing a recombinant nucleoprotein derived from influenza viruses subtype A H1N1 (available from Sino Biological Company, trade name: 11675-V08H) was prepared at a concentration of 500 micrograms/milliliter.
Similarly, four solutions containing recombinant nucleoproteins derived from influenza viruses subtype A H1N1, H2N2, H3N2, and H7N9 (available from Sino Biological Company, trade name: 40205-V08H, 40033-V08H, 40208-V08H, and 40111-V08H) were prepared at a concentration of 500 micrograms/milliliter, respectively.
Furthermore, a solution containing a recombinant nucleoprotein (available from ORLA) derived from influenza viruses subtype B was prepared at a concentration of 500 micrograms/milliliter. Hereinafter, the six solutions are referred to as “Solution group A”.
a part of each of the six solutions included in the solution group A was diluted 50 fold with a PBS containing both of 5% skim milk (available from Wako Pure Chemical Industries Ltd.) and 0.05% of tween 20 (hereinafter, this PBS is referred to as “skim-milk-containing PBST”). In this way, a diluted solution group B (Concentration: 10 micrograms/milliliter) including six diluted solutions of the recombinant NP was obtained.
Apart of each of the six solutions included in the diluted solution group B was diluted 10-fold again with the skim-milk-containing PBST. In this way, a diluted solution group C (Concentration: 1 microgram/milliliter) including six diluted solutions of the recombinant NP was obtained. This was repeated to obtain a diluted solution group D (Concentration: 0.1 microgram/milliliter), a diluted solution group E (Concentration: 0.01 microgram/milliliter), a diluted solution group F (Concentration: 0.001 microgram/milliliter), a diluted solution group G (Concentration: 1×10−4 microgram/milliliter), and a diluted solution group H (Concentration: 1×10−5 microgram/milliliter).
The solution containing the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 (concentration: 5 micrograms/milliliter) was injected to the wells of 96-well microplate (Maxisorp, Nunc). Each of the wells contained 200 microliters of the solution. The 96-well plate was left at rest at room temperature for two hours to immobilize the virus in the wells.
The skim-milk-containing PBST was injected in each well to block the virus. The volume of the PBST injected to each well was 250 microliters. The 96-well plate was left at rest at room temperature for three hours.
PBST containing 0.05% tween 20 was injected in each well to wash the wells. The PBST had a pH of 7.4. The volume of the PBS injected to each well was 200 microliters. This was repeated twice.
Each of the diluted solutions of the recombinant NP included in the diluted solution groups B-H were injected into each well. As a reference, the skim-milk-containing PBST was injected to another well. This well including the skim-milk-containing PBST only was used as a reference to remove a background upon measurement. The volume of the solutions injected in each well was 100 microliters. The 96-well plate was left at rest at room temperature. In this way, the recombinant NPs contained in the solutions B-G was bound to the anti-NP VHH antibodies contained in the wells. The 96-well plate was left at rest at room temperature for one hour.
PBS containing 0.05% tween 20 was injected in each well to wash the wells. The PBS had a pH of 7.4. The volume of the PBS injected to each well was 200 microliters. This was repeated five times.
The anti-NP antibody (available from abcam company, trade name: ab110661) was diluted with the PBST containing 0.05% tween 20 (concentration: 5 micrograms/milliliter), and injected to each well. The volume of the PBST injected to each well was 100 microliters. In this way, the anti-NP antibody was bound to the recombinant NPs contained in the wells. The 96-well plate was left at rest at room temperature for one hour.
PBS containing 0.05% tween 20 was injected in each well to wash the wells. The PBS had a pH of 7.4. The volume of the PBS injected to each well was 200 microliters. This was repeated five times.
Labelled antibodies (available from Santa Cruz company, trade name: goat anti-mouse IgG-HRP) were diluted 20,000 fold with PBST containing 0.05% tween 20. The thus-diluted labelled antibodies was inject to each well (50 microliters/well). Then, the 96-well plate was left at rest for one hour.
PBS containing 0.05% tween 20 was injected in each well to wash the wells. The PBS had a pH of 7.4. The volume of the PBS injected to each well was 200 microliters. This was repeated five times.
The color-producting agent (available from Thermo Scientific, trade name: 1-step ultraTMB-ELISA) was injected into each well (50 microliters/well). The 96-well plate was left at rest for thirty minutes to cause the color-producting agent to react with the antibody.
The color-stopping agent (available from ScyTek laboratories, trade name: TMB Stop Buffer) containing sulfuric acid and hydrochloric acid at a low concentration was injected into each well at a concentration of 50 microliters/well to cease the reaction.
The absorbance of the solution at a wavelength of 450 nanometers was measured. FIGS. 4A-4F are graphs showing the measurement result of the cross reactivity of the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 with regard to the influenza virus type A H1N1 11675, H1N1 40205, H2N2, H3N2, H7N9, and the influenza virus type B, respectively.
As understood from FIGS. 4A-4F, the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 has high cross reactivity to the recombinant intranuclear proteins derived from influenza viruses type A H1N1, H2N2, H3N2, H7N9. On the other hand, the VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 08 has low cross reactivity to the influenza virus type B.
INDUSTRIAL APPLICABILITY
The present invention provides a composite comprising a novel antibody capable of binding to the intranuclear protein of the influenza virus. The present invention also provides a detection device and a detection method using the composite comprising the novel antibody.

Claims (8)

The invention claimed is:
1. A composite, comprising:
an antibody; and
at least one selected from the group consisting of a solid phase support and a labeled substance,
wherein
the antibody consists of the amino acid sequence represented by SEQ ID NO: 08, and is capable of binding to an intranuclear protein of an influenza virus type A;
the influenza virus type A is at least one selected from the group consisting of H1N1, H2N2, H3N2, and H7N9; and
the antibody is bound to the at least one selected from the group consisting of the solid phase support and the labeled substance.
2. The composite according to claim 1, wherein
the composite comprises the solid phase support; and
the solid phase support is selected from the group consisting of a plate, a bead, a disk, a tube, a filter, and a film.
3. The composite according to claim 1, wherein
the composite comprises the labeled substance; and
the labeled substance is selected from the group consisting of a fluorescent substance, a luminescent substance, a dye, and a radioactive substance.
4. A detection device, comprising:
a composite; and
a detector;
wherein
the composite comprises an antibody and at least one selected from the group consisting of a solid phase support and a labeled substance;
the antibody consists of the amino acid sequence represented by SEQ ID NO: 08, and is capable of binding to an intranuclear protein of an influenza virus type A;
the influenza virus type A is at least one selected from the group consisting of H1N1, H2N2, H3N2, and H7N9;
the antibody is bound to the at least one selected from the group consisting of the solid phase support and the labeled substance; and
the detector detects an antigen-antibody reaction of the antibody and the intranuclear protein of the influenza virus type A which is contained in an analyte.
5. A detection method, comprising:
(a) bringing a composite into contact with an analyte;
wherein
the composite comprises an antibody and at least one selected from the group consisting of a solid phase support and a labeled substance;
the antibody consists of the amino acid sequence represented by SEQ ID NO: 08, and is capable of binding to an intranuclear protein of an influenza virus type A;
the influenza virus type A is at least one selected from the group consisting of H1N1, H2N2, H3N2, and H7N9; and
the antibody is bound to the at least one selected from the group consisting of a solid phase support and the labeled substance; and
(b) detecting an antigen-antibody reaction of the antibody and the intranuclear protein of the influenza virus type A which is contained in an analyte.
6. A composite comprising:
an antibody consisting of an amino acid sequence; and
at least one selected from the group consisting of a solid phase support and a labeled substance,
wherein
the amino acid sequence consists of, in an N- to C-direction, the following structural domains:

N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C
wherein
FR denotes a framework region amino acid sequence and CDR denotes a complementary determining region amino acid sequence;
the CDR1 consists of an amino acid sequence represented by SEQ ID NO: 01;
the CDR2 consists of an amino acid sequence represented by SEQ ID NO: 02;
the CDR3 consists of an amino acid sequence represented by SEQ ID NO: 03;
the FR1 consists of an amino acid sequence represented by SEQ ID NO: 04;
the FR2 consists of an amino acid sequence represented by SEQ ID NO: 05;
the FR3 consists of an amino acid sequence represented by SEQ ID NO: 06;
the FR4 consists of an amino acid sequence represented by SEQ ID NO: 07; and
the antibody is bound to the at least one selected from the group consisting of the solid phase support and the labeled substance, and is capable of binding to an intranuclear protein of an influenza virus type A.
7. The detection device according to claim 4, wherein the detector detects a change of a physical amount based on the antigen-antibody reaction of the antibody and the intranuclear protein of the influenza virus type A which is contained in the analyte; and
wherein the physical amount is luminescence intensity, chromaticity, light transmission, turbidness, absorbance, or radiation dose.
8. The detection method according to claim 5, comprising detecting a change of a physical amount based on the antigen-antibody reaction of the antibody and the intranuclear protein of the influenza virus type A which is contained in the analyte;
wherein the physical amount is luminescence intensity, chromaticity, light transmission, turbidness, absorbance, or radiation dose.
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